Application of circ0005704 and related biological elements thereof in diagnosis and treatment of recurrent spontaneous abortion with unknown reasons

Abstract

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to application of circ0005704 and related biological elements thereof in diagnosis and treatment of recurrent spontaneous abortion with unknown reasons. Researches find that differential expression and autophagy level of ULK1 in trophoblast cells of URSA patients are increased. High-throughput transcriptome sequencing is combined with bioinformatics analysis, and it is found that miR-26a-5p is down-regulated in URSA patients, is responsible for up-regulation of ULK1 and promotes autophagy of trophoblast cells, so that occurrence of URSA is increased. Through a high-throughput transcriptional set screening strategy and dual luciferase reporter gene analysis, it is found that circ0005704 enhances the expression of ULK1 through miR-26a-5p. In a word, the research of the invention shows that the circ0005704 / miR-26a-5p / ULK1 signal axis participates in the pathogenesis of URSA by adjusting the migration of the trophoblast cells, and a new target and scientific evidence are provided for the clinical treatment of URSA.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and molecular biology, specifically relating to the application of circ_0005704 and its related biological elements in the diagnosis and treatment of unexplained recurrent spontaneous abortion. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Unexplained recurrent spontaneous abortion (URSA) is defined as two or more consecutive spontaneous abortions with the same partner before 20 weeks of gestation. URSA is the most common complication of pregnancy, with an incidence of up to 5% in women of reproductive age. The risk of recurrent miscarriage increases significantly with the number of miscarriages. Despite the achievements of researchers worldwide in studying the pathogenesis of RSA, approximately 50% of RSA cases remain unexplained, aside from known causative factors such as genetics, uterine abnormalities, thrombosis, hormonal or metabolic disorders, infection, autoimmune factors, and abnormal sperm parameters. Recurrent pregnancy failure causes severe physical harm to patients and places enormous stress on families. Therefore, exploring the pathogenesis, identifying intervention targets and regulatory mechanisms, and elucidating proven effective drugs or methods are urgent issues in the field of reproductive medicine.

[0004] The beginning of new life begins with the fusion of an egg and sperm. After fertilization, the fertilized egg undergoes several rounds of division and morphogenesis, gradually forming a blastocyst. At this stage, the blastocyst cells have differentiated into two parts: the trophoblast and the inner cell mass, preparing for implantation. The key to successful embryo implantation lies in the synchronous differentiation and development of the trophoblast and endometrial receptivity, as well as the mutual recognition and communication between the blastocyst and the endometrium. Trophoblast cells penetrate the luminal epithelium, cross the basement membrane, and embed themselves in the matrix of the uterine wall. Trophoblast cells are fetal cells that directly interact with the maternal system and play a crucial role in embryo implantation and the formation of a functional placenta. Therefore, disruption of trophoblast function can lead to a range of adverse pregnancy outcomes, such as malformations, fetal growth restriction, miscarriage, and stillbirth. Currently, the role and regulatory mechanisms of trophoblast dysfunction in URSA are unclear. Therefore, elucidating the function and regulatory mechanisms of trophoblast cells will provide new targets and insights for the clinical treatment of URSA.

[0005] Autophagy is a major catabolic process in eukaryotic cells, responsible for the degradation and recycling of damaged macromolecules and organelles. It has become a hot research topic in the medical field. Recent studies have shown that autophagy dysfunction is associated with the development of various diseases, such as tumors, neurodegenerative diseases, and metabolic disorders. Furthermore, autophagy is considered to potentially provide new pathways and targets for disease prevention and treatment. While autophagy is primarily a cellular defense mechanism, excessive autophagic degradation can be harmful in many cases. Under specific conditions, when ULK1 is activated, it triggers the activation of the beclin1-containing phosphoinositol 3-kinase (PI3K) complex, initiating autophagosome formation. Therefore, ULK1 is a key regulator of autophagy, and changes in its expression affect autophagosome formation and function. It has been found that excessive activation of trophoblastic autophagy promotes URSA. For example, Cai et al. found that decreased MFN2 expression levels lead to mitochondrial dysfunction, increased autophagy, and impaired trophoblastic cell function, potentially leading to early unexplained miscarriage; Pan et al. revealed that damage to Shh signaling triggers autophagy, thereby inhibiting trophoblastic migration in recurrent miscarriage. However, the role of ULK1 in regulating trophoblast autophagy during URSA has not been fully elucidated. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide the application of circ_0005704 / miR-26a-5p in the diagnosis and treatment of unexplained recurrent spontaneous abortion (URSA). Specifically, this invention has discovered increased differential expression of ULK1 and autophagy levels in trophoblast cells of URSA patients. Combining high-throughput transcriptome sequencing with bioinformatics analysis revealed that miR-26a-5p is downregulated in URSA patients and is responsible for upregulating ULK1, promoting trophoblast cell autophagy, thereby increasing the incidence of URSA. Through high-throughput transcriptome screening and dual-luciferase reporter gene analysis, it was found that circ_0005704 enhances ULK1 expression through miR-26a-5p. In summary, the research of this invention demonstrates that the circ_0005704 / miR-26a-5p / ULK1 signaling axis participates in the pathogenesis of URSA by regulating trophoblast cell migration, providing new targets and scientific evidence for the clinical treatment of URSA.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] In a first aspect, the invention provides the use of substances that detect the expression levels of circ_0005704 and its related biological elements in the preparation of products for screening, diagnosis, detection, monitoring, or prognosis of unexplained recurrent spontaneous abortion.

[0009] The bio-elements associated with circ_0005704 include miR-26a-5p and ULK1.

[0010] Specifically, this invention found that circ_0005704 promotes autophagy in trophoblast cells, thereby determining migration and miscarriage rates in URSA patients. Overexpression of circ_0005704 eliminates the inhibitory effect of miR-26a-5p on ULK1, leading to its accumulation and enhanced autophagy, thus affecting trophoblast cell migration. Mechanistically, circ_0005704 acts as a molecular sponge for miR-26a-5p, thereby weakening the binding of miR-26a-5p to the 3'UTR of ULK1 mRNA. Importantly, upregulation of circ_0005704 is closely associated with downregulation of miR-26a-5p and is positively correlated with ULK1-mediated autophagy in patients and URSA models. Therefore, circ_0005704 and its related biofactors can serve as (auxiliary) diagnostic and prognostic biomarkers for unexplained recurrent spontaneous abortion.

[0011] The substances used to detect the expression levels of circ_0005704 and its related biological elements may include reagents for detecting the expression levels of circ_0005704, miR-26a-5p, and ULK1 encoding genes based on real-time quantitative PCR, in situ hybridization, gene chips, and gene sequencing, and / or reagents for detecting the expression level of ULK1 protein based on immunoassay methods.

[0012] The products mentioned include, but are not limited to, primers, probes, (gene or protein) chips, nucleic acid membrane strips, detection kits, detection devices, and detection equipment for detecting the expression levels of circ_0005704 and its related biological elements, without specific limitations herein.

[0013] A second aspect of the present invention provides a system for screening, diagnosing, detecting, monitoring, or predicting unexplained recurrent spontaneous abortion, the system comprising:

[0014] The acquisition unit is configured to acquire the expression levels of circ_0005704 and its related biological elements in the subject's test sample;

[0015] The assessment unit is configured to assess and determine the subject's disease status based on the subject's circ_0005704 and its related bio-elements obtained by the acquisition unit.

[0016] The bio-elements associated with circ_0005704 include miR-26a-5p and ULK1.

[0017] The test sample can be a blood sample (including peripheral blood mononuclear cells) or a villous tissue sample from the subject.

[0018] A third aspect of the invention provides the use of circ_0005704 and related biological elements as targets in the preparation and / or screening of drugs for unexplained recurrent spontaneous abortion.

[0019] Specifically, the effects of candidate drugs on circ_0005704 and its related biological elements before and after use can be used to determine whether candidate drugs can be used to prevent or treat unexplained recurrent spontaneous abortion.

[0020] Specifically, methods for screening medications to prevent or treat unexplained recurrent spontaneous abortion include:

[0021] 1) Treat systems expressing and / or containing circ_0005704 and its related biological elements with candidate substances; set up parallel controls without candidate substance treatment;

[0022] 2) After completing step 1), detect the expression level of circ_0005704 and its related biological elements in the system; compared with the parallel control, if the expression level of circ_0005704 and its related biological elements in the system treated with the candidate substance is significantly downregulated or upregulated, the candidate substance can be used as a candidate drug for the prevention or treatment of unexplained recurrent spontaneous abortion.

[0023] The bio-elements associated with circ_0005704 include miR-26a-5p and ULK1.

[0024] More specifically, step 2) is as follows: if the expression of circ_0005704 is significantly downregulated, the expression of miR-26a-5p is significantly upregulated, and / or the expression of ULK1 is significantly downregulated in the system treated with the candidate substance, then the candidate substance can be used as a candidate drug for the prevention or treatment of unexplained recurrent spontaneous abortion.

[0025] The system may be a cellular system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system, without any specific limitation.

[0026] A fourth aspect of the invention provides the use of a substance that inhibits or reduces the expression of circ_0005704 in any one or more of the following:

[0027] a) Promote miR-26a-5p expression or prepare products that promote miR-26a-5p expression;

[0028] b) Inhibit ULK1 expression or prepare products that inhibit ULK1 expression;

[0029] c) Promote the migration of trophoblast cells or prepare products that promote the migration of trophoblast cells;

[0030] d) Inhibit autophagy in trophoblast cells or prepare products that inhibit autophagy in trophoblast cells;

[0031] g) Products for the prevention and / or treatment of unexplained recurrent spontaneous abortion.

[0032] Among them, substances that inhibit the expression of circ_0005704 or reduce its activity can be, but are not limited to, RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA (SEQ ID NO.7-8), shRNA, substances that carry out lentiviral infection or gene knockout, and are not specifically limited here.

[0033] The product can be a drug or a laboratory reagent for non-pharmaceutical purposes, and the laboratory reagent can be used for basic research. For example, the product can be used to regulate the migration and autophagy of trophoblast cells in vitro, thereby preparing trophoblast cell-related biological models, thus providing convenient conditions for the study of the mechanisms of diseases such as unexplained recurrent spontaneous abortion.

[0034] According to the present invention, when the product is a drug, the drug further includes at least one inactive pharmaceutical ingredient.

[0035] The inactive pharmaceutical ingredient may be a carrier, excipient, or diluent commonly used in pharmaceuticals. Furthermore, the carriers, excipients, and diluents that may be included are well-known in the art, and those skilled in the art can determine that they meet clinical standards.

[0036] The medicament of the present invention can be administered into the body by known means. For example, it can be delivered to the tissue of interest via intravenous systemic delivery or local injection. Alternatively, it can be administered via intravenous, percutaneous, intranasal, mucosal, or other delivery methods. Such administration can be performed via single or multiple doses. It will be understood by those skilled in the art that the actual dose to be administered in the present invention can vary considerably depending on a variety of factors, such as the target cells, biological type or tissue thereof, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0037] The drug can be administered to humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, and chimpanzees, without any specific limitation.

[0038] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0039] The above-described technical approach demonstrates that circ_0005704 promotes autophagy in trophoblast cells, thereby determining migration and miscarriage rates in URSA patients. Overexpression of circ_0005704 eliminates the inhibitory effect of miR-26a-5p on ULK1, leading to its accumulation and enhanced autophagy, thus affecting trophoblast cell migration. Mechanistically, circ_0005704 acts as a molecular sponge for miR-26a-5p, thereby weakening the binding of miR-26a-5p to the 3'UTR of ULK1 mRNA. Importantly, the upregulation of circ_0005704 is closely associated with the downregulation of miR-26a-5p and is positively correlated with ULK1-mediated autophagy in patients and URSA models. In summary, the above-described technical approach reveals the regulatory axis of ULK1-mediated autophagy to promote the pathogenesis of URSA caused by dysregulation of circ_0005704 and miR-26a-5p, thus possessing significant potential for practical application. Attached Figure Description

[0040] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0041] Figure 1 To illustrate the upregulation of autophagy and its association with the pathogenesis of URSA in this embodiment of the invention: (A) Detection of autophagosome accumulation in villous tissue of NP subjects and URSA patients using electron microscopy (scale bar, 50 μm). (B) Determination of ULK1, Beclin1, ATG7, ATG13, LC3, and p62 protein levels in villous tissue of NP subjects and URSA patients (n=30) using Western blotting. (C) Representative IF staining images of ULK1 in villous tissue of NP subjects and URSA patients (scale bar, 50 μm). (D) qRT-PCR analysis of ULK1 expression in villous tissue of NP subjects (n=30) and URSA patients (n=10). (E) Evaluation of the diagnostic value of ULK1 in villous tissue of NP subjects and URSA patients using ROC curves (n=30). (FG) A URSA mouse model was established. The day the vaginal plug was discovered was designated as day 0.5 of gestation. Each female mouse was intravenously injected with DMSO, CQ, and SBI-0206965 at a dose of 25 mg / kg / day via the tail vein and sacrificed on day 11.5 of gestation. The embryo resorption rate of the four groups of treated URSA mice was calculated, with arrows indicating resorbed embryos (n=5). (H) The protein levels of ULK1, LC3, and P62 were measured in the placenta of pregnant mice (n=5). Values ​​are listed as mean ± SD. *P<0.05, ****P<0.0001, ns: no statistical significance.

[0042] Figure 2 In this embodiment of the invention, miR-26a-5p expression was negatively correlated with ULK1 expression in URSA patients; (A) Microarray analysis confirmed reduced expression of nine miRNAs in PBMCs of NP subjects and URSA patients (P<0.05, fold change>1.5). Red dots indicate increased expression, while green dots indicate decreased expression (n=3). (B) Venn plots show microRNAs predicted by Target Scan, miRDB, DIANA, and Chip (P<0.05, fold change>1.5). (C) qRT-PCR analysis of miR-26a-5p expression in villous tissue of NP subjects and URSA patients (n=30). (D) FISH analysis of the location and expression level of miR-26a-5p in villous tissue (miR-26a-5p, red; DAPI, blue; scale bar = 50 μm). (E) ROC curves were used to evaluate the diagnostic value of miR-26a-5p for URSA (n=30). (F) The correlation between miR-26a-5p and ULK1 mRNA in URSA villous tissue was analyzed by Pearson correlation analysis (n=30). Values ​​are listed as mean ± SD. ****P<0.0001.

[0043] Figure 3 This invention illustrates the following: (A) A schematic diagram of the putative binding sequence of miR-26a-5p in the 3'UTR of ULK1 mRNA, showing the luciferase activity of wild-type (WT) and mutant (MUT) constructs. (B) Luciferase activity was measured by co-transfecting vectors (ULK1 3′UTR-WT, MUT1, MUT2, MUT1+MUT2) with a negative control (NC) and a miR-26a-5p mimic into 293T cells. (CD) qRT-PCR analysis of miR-26a-5p and ULK1 mRNA expression in HTR-8 / Svneo cells transfected with NC, a miR-26a-5p mimic, INC, and a miR-26a-5p inhibitor. (E) ULK1, LC3, and P62 protein levels were measured in HTR-8 / Svneo cells after transfection with NC, miR-26a-5p mimic, INC, and miR-26a-5g inhibitors. Values ​​are listed as mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001; ns: no statistical significance.

[0044] Figure 4(A) In this embodiment of the invention, circ_0005704 interacts with miR-26a-5p expression and shows a negative correlation; (B) Microarray analysis identifies 24 predicted potential circ-RNAs that may target miR-26a-5p, screened by PBMCs of miRanda v3.3a and NP subjects and URSA patients (P<0.05, fold change>1.5). Red dots indicate increased expression, while green dots indicate decreased expression (n=3). (C) RNA pull-down assay by qRT-PCR shows the binding of circ-RNAs to miR-26a-5p via the Bio probe. (D) Relative expression of GAPDH, U6, circ_0005704, and circ_0064954 in the nucleus and cytoplasm, respectively. (E) RIP analysis by qRT-PCR showed enrichment of AGO2-downregulated circ_0005704 in miR-26a-5p mimics treated with HTR-8 / Svneo. (F) RNA pull-down assay by qRT-PCR showed enrichment of miR-26a-5p pulled down by the circ_0005704 probe in HTR-8 / Svneo. (G) qRT-PCR analysis detected the expression of circ_0005704 in trophoblast cells after RNase R treatment. (H) qRT-PCR detection of circ_0005704 expression in villous tissue of NP group and URSA patients (n=30). (I) ROC curves (n=30) were used to evaluate the diagnostic value of circ_0005704 for URSA. (J) The correlation between miR-26a-5p and circ_0005704 in villous tissue was analyzed by Pearson correlation analysis (n=30). (K) A schematic diagram of the miR-26a-5p binding sequence in the circ_0005704 sequence was predicted using bioinformatics software. Wild-type (WT) and mutant (MUT) cells were constructed, and luciferase activity was measured. (L) Luciferase activity was measured by co-transfecting 293T cells with the vector (circ_0005704-WT, MUT) along with a negative control (NC) and a miR-26a-5p mimic. Values ​​are mean ± SD. *P<0.05, **P<0.01, ***P<0.001; ns: no statistical significance.

[0045] Figure 5In this embodiment of the invention, HTR-8 / Svneo cells were transfected with a control vector plasmid, the circ_0005704 plasmid, or a mixture of the circ_0005704 plasmid and miR-26a-5p mimic. Overexpression of circ_0005704 promoted autophagy through the miR-26a-5p / ULK1 axis. An NP mouse model was constructed, and the vaginal plugs of the mice were examined every morning. The day on which the vaginal plug was visible was designated as day 0.5 of gestation. On days 0.5, 3.5, 6.5, and 9.5, female mice were intravenously injected with the circ_0005704 plasmid at a dose of 320 ng / g / d using the in vivo transfection reagent Entranster™ (Engreen, Beijing, China), or simultaneously injected intravenously with a mixture of the circ_0005704 plasmid at a dose of 320 ng / g / d and 10 nmol / d miR-26a-5p mimic. Mice were sacrificed on day 11.5 of gestation. (AC) Relative expression of circ_0005704, miR-26a-5p, and ULK1 mRNA in HTR-8 / Svneo cells. (D) Measurement of ULK1, LC3, and P62 protein levels in HTR-8 / Svneo cells. (E) Representative micrographs showing HTR8 / Svneo cells fixed and stained on the basal side of the invasion membrane. (F) Determine the expression levels of MMP9, E-cadherin, N-cadherin, and vimentin in HTR-8 / Svneo cells. (GH) Calculate the embryo resorption rate in three groups of URSA mice; arrows indicate resorbed embryos (n=5). (I) Assess the expression levels of ULK1, LC3, and P62 proteins in the placenta of NP-pregnant mice. (J) Determine the expression of MMP9, E-cadherin, N-cadherin, and vimentin in the placenta of NP-pregnant mice. Values ​​are listed as mean ± SD. ***P<0.001, ****P<0.0001.

[0046] Figure 6In this embodiment of the invention, circ_0005704 knockdown inhibits autophagy and reduces embryonic resorption via miR-26a-5p / ULK1; HTR-8 / Svneo cells were transfected with si-NC, si-circ_0005704, si-circ_0005704, and miR-26a-5p inhibitors. A URSA mouse model was constructed, and vaginal plugs in mice were examined every morning; the day a vaginal plug was visible was designated as day 0.5 of gestation. URSA mice were transfected via tail vein injection at days 0.5, 3.5, 6.5, and 9.5 using the in vivo transfection reagent Entranster™ (Engreen, Beijing, China). The transfection doses were 10 nmol si-NC, 10 nmol si-circ_0005704, or a mixture of 10 nmol si-circ_0005704 and 10 nmol miR-26a-5p inhibitors. Mice were sacrificed on day 11.5 of gestation. (AC) Relative expression of circ_0005704, miR-26a-5p, and ULK1 mRNA in HTR-8 / Svneo cells. (D) Measurement of ULK1, LC3, and P62 protein levels in HTR-8 / Svneo cells. (E) Representative micrographs showing HTR8 / Svneo cells fixed and stained on the basal side of the invasion membrane. (F) Determine the expression levels of MMP9, E-cadherin, N-cadherin, and vimentin in HTR-8 / Svneo cells. (GH) Calculate the embryo resorption rate in the three groups of URSA mice; arrows indicate resorbed embryos (n=5). (I) Measure the expression levels of ULK1, LC3, and P62 proteins in the placenta of URSA pregnant mice. (J) Determine the expression of MMP9, E-cadherin, N-cadherin, and vimentin in the placenta of URSA pregnant mice. Values ​​are listed as mean ± SD. *P<0.05, **P<0.01, ***P<0.001. Detailed Implementation

[0047] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0048] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0049] The present invention will now be further illustrated with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified, experimental conditions not explicitly stated in the examples are generally performed under conventional conditions or as recommended by the reagent company. Unless otherwise specified, all reagents and consumables used in the following examples are commercially available.

[0050] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. The following examples illustrate experimental methods under specific conditions, which are generally performed according to conventional conditions. The nucleotide sequences used in the examples are shown in the table below:

[0051]

[0052]

[0053] Example

[0054] 1. Materials and Methods

[0055] 1.1 Patients

[0056] From September 2019 to December 2023, we recruited 30 women with URSA and 30 women with normal pregnancies (NP) from the Reproductive Medicine Center of the 960th Hospital and Qianfoshan Hospital for this study. In this study, all risk factors with known etiologies, such as chromosomal abnormalities, maternal reproductive tract abnormalities, endocrine abnormalities, immune dysfunction, reproductive tract infections, cervical insufficiency, and thrombosis, were excluded. Written consent and confidentiality agreements were obtained for all clinical specimens collected.

[0057] 1.2 Specimen Collection

[0058] Peripheral blood and chorionic villus tissue were obtained from URSA patients and NP women, respectively. Peripheral blood was collected using blood collection tubes containing ethylenediaminetetraacetic acid (EDTA) (Becton Dickinson), followed by separation of peripheral blood mononuclear cells (PBMCs) by Ficoll density gradient centrifugation. The separated plasma was frozen and stored at -80°C and used for further studies with the PBMCs. Regarding chorionic villus tissue collection, fresh chorionic villus tissue was obtained after termination of pregnancy via uterine dilation and aspiration. A portion of the tissue was fixed in 4% formaldehyde for subsequent immunofluorescence and FISH experiments. The remaining chorionic villus tissue was frozen and stored at -80°C for Western blotting (WB) and polymerase chain reaction (PCR) analysis.

[0059] 1.3 Transmission Electron Microscopy

[0060] Fresh human chorionic villus tissue was cut into pieces with a volume of approximately 1 mm. 3 Small pieces of the tissue were fixed in 2.5% glutaraldehyde at room temperature. The fixed tissue was then rinsed with phosphate-buffered saline (PBS) and further fixed in 1% osmium tetroxide at 4°C for 2 hours. The tissue was then dehydrated, embedded, and polymerized. Fourth, the embedded tissue was sectioned to a thickness of 70 nm. Fifth, the prepared sections were stained with 3% uranium acetate saturated alcohol solution for 8 minutes, followed by staining with 2.7% lead acetate solution for 8 hours. The distribution and number of autophagosomes were observed using transmission electron microscopy.

[0061] 1.4 Immunofluorescence (IF)

[0062] Immunofluorescence was used to detect the expression and distribution of ULK1 in NP and URSA villous tissues. First, fresh villous tissue was fixed with 4% paraformaldehyde at room temperature and then prepared into 5 μm paraffin sections. Second, the sections were dried in a sliding desiccator at 65°C for 60 min. Third, the sections were dehydrated in graded alcohols and subjected to antigen retrieval for 20 min. Fourth, endogenous peroxidase was removed, and the sections were blocked with serum for 30 min. Fifth, the sections were incubated with ULK1 (8054, CST) overnight at 4°C. The next day, the primary antibody was bound to FITC mouse anti-rabbit antibody. Finally, the cell nuclei were stained with 6-diamino-2-phenylindole (DAPI), and images were captured using a microscope (BZ-X800LE, Keyence, Japan).

[0063] 1.5 Fluorescence in situ hybridization (FISH)

[0064] The localization and expression of miR-26a-5p in villous tissue from NP and URSA patients were detected using FISH assays. The results showed that it could clearly distinguish between NP and URSA. The preparation of villous tissue sections was the same as that for immunofluorescence. First, the sections were incubated with a diluted proteinase K solution at 37°C for 20 minutes. Then, the sections were dehydrated with a gradient of alcohols. Afterward, they were incubated with a denaturing agent in a 78°C oven for 8 minutes. Next, the sections were incubated with Cy3-labeled miR-26a-5p probes in the dark at 37°C for 16 hours. Finally, the cell nuclei were stained with DAPI working solution and then incubated in the dark for 10 minutes. The samples were then observed and photographed under a microscope. All procedures were performed according to the manufacturer's protocol (Genepharma, Shanghai, China).

[0065] 1.6 Microarray Analysis

[0066] Total RNA was extracted from PBMCs using TRIzol (Invitrogen, Carlsbad, CA, USA). OE Biotech (Shanghai, China) conducted trials on URSA patients (n=3) and control subjects (n=3). Sample labeling, microarray hybridization, and washing were performed according to the manufacturer's standard protocol. In this article, the top 19 downregulated miRNAs with a fold change >2 (P<0.05) were selected as candidates for further research. Furthermore, circular RNAs were investigated in depth to gain a more comprehensive understanding of their mechanisms of action.

[0067] 1.7 Cell Culture and Treatment

[0068] 293T cells and the human feeder cell line HTR-8 / Svneo were obtained from the Cell Bank of the Chinese Academy of Sciences. All cells were cultured in 1640 (Bioind, Kibbiz, Israel) containing 10% FBS (Bioind, Kibbiz, Israel) and 1% penicillin / streptomycin. Cells were maintained in an incubator at 37°C with 5% CO2.

[0069] 1.8 Cell Transfection

[0070] Cells containing circ_0005704, vevtor, miR-26a-5p mimics, negative control (NC), small interference negative control (siNC), si-circ_0005704, miR-26a-5p inhibitor, and inhibitor negative control (INC) were purchased from GenePharma (Shanghai, China). Transfection was performed using lipofectamine 2000 (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. First, cells were loaded at 1 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of 100 cells / mL into 12-well plates. After reaching 50% confluence, the cells were transfected with Lipofectamine 2000 reagent. After 48 hours, the cells were used for further experiments.

[0071] 1.9 Dual-luciferase reporter gene assay

[0072] The 3'UTR region of ULK1 containing the miR-26a-5p binding or mutant sequence and circ_0005704 were cloned into the pmirGLO luciferase vector (Promega, USA) to synthesize wild-type plasmids (ULK1-WT and circ_0005704-WT) or mutant plasmids (ULK1-MUT and circ_0005704-MUT). These plasmids, along with NC mimics or miR-26a-5p, were transfected into 293T cells using Lipofectamine 2000 (Invitrogen). Forty-eight hours later, luciferase activity was measured using a dual luciferase reporter assay kit (Promega, USA) and normalized to ranila luciferase activity.

[0073] 1.10 RNA Immunoprecipitation Protein (RIP)

[0074] RIP assays were performed using the PureBinding RNA Immunoprecipitation Kit (Geneseed, P0102, Guangzhou) according to the manufacturer's instructions. A total of 1×10⁻⁶ samples were harvested. 7 HTR8 sneo cells were lysed in RIP lysis buffer, and the supernatant was collected after centrifugation at 12000g for 15 minutes. Simultaneously, proteins A and G were incubated with anti-IgG (2729S, Cell Signaling Technology) or anti-AGO2 (ab186733, Abcam) at 4°C for 2 hours and washed with lysis buffer. The supernatant was then incubated with proteins A and G overnight at 4°C. RNA was extracted from the immunoprecipitate, and the expression of miRNA and circRNA was detected by qRT-PCR using specific primers.

[0075] 1.11 RNA pull-down

[0076] Biotin-labeled circ-RNA probes were provided by Invitrogen, and RNA was pulled down according to the instructions of the Magnetic RNA Protein Pull-Down Kit (Thermo Fisher Scientific, USA). In short, HTR-8 / Svneo cell RNA extracts were incubated with 2 μg of the probe and 40 μL of streptavidin beads at 4°C for 2 hours. After centrifugation, the RNA complexes were collected, washed, and analyzed by qRT-PCR.

[0077] 1.12 Transwell assay

[0078] Pretreated cells were resuspended in serum-free medium to prepare cells with a density of 2 × 10⁶ cells / mL. 5Single-cell suspensions were prepared, and 700 μL of serum-containing medium was added to the lower chamber. After culturing for 24 hours, cells that had not migrated from the upper chamber were removed. Cells that had migrated out of the chamber were fixed with 4% paraformaldehyde for 30 minutes and washed three times with PBS for 5 minutes each time. Then, the fixed cells were stained with crystal violet for 10 minutes and washed appropriately with PBS. Finally, the number of migrated cells was observed and counted under a microscope and recorded with photographs.

[0079] 1.13 Subcellular localization

[0080] RNA was extracted and isolated from the nucleus and cytoplasm of HTR-8 / Svneo cells using a cytoplasmic and nuclear RNA purification kit (Amyjet Scientific, China). First, cells were lysed using cell lysis buffer, and the supernatant containing cytoplasm was clearly separated from the precipitated nuclei by centrifugation. Then, cytoplasmic RNA and nuclear RNA were purified separately, and the expression levels of circ_0005704 in the nucleus and cytoplasm were determined by qRT-PCR to identify its subcellular localization. U6 and GAPDH were considered positive controls for the nucleus and cytoplasm, respectively.

[0081] 1.14 RNA isolation and quantitative real-time PCR (qRT-PCR)

[0082] Total RNA was extracted using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. RNA was reverse transcribed using the miRNA first-strand cDNA synthesis kit (Vazyme, Nanjing, China) or the PrimeScript RT kit for mRNA (Toyobo, Osaka, Japan) according to the manufacturer's instructions. qRT-PCR was performed using SYBR Green (Invitrogen, Waltham, MA, USA) on an Applied Biosystems 7500 instrument (Applied Biosystems, Foster, USA) for miRNA and mRNA analysis, and primer sequences are displayed. Triple replicates were set up for each sample. Comparisons were made using 2... -ΔΔCt The method involved relative RNA quantification. The relative expression levels of miRNAs were normalized to the level of the internal control U6, while the relative expression levels of genes were normalized to the expression level of ACTB in each sample.

[0083] 1.15 Western blot analysis

[0084] Cells and villous tissues were lysed in RIPA buffer (Thermo Scientific, Massachusetts, USA) with protease and phosphatase inhibitors (Thermo Scientific, Massachusetts, USA), and proteins were analyzed by Western blotting as previously described. Protein concentrations were determined using the BCA Protein Assay Kit (Thermo Scientific, Massachusetts, USA) according to the manufacturer's instructions. Anti-ULK1 antibody (8054, Cell Signaling Technology), ATG7 antibody (8558s, Cell Signaling Technology), ATG13 antibody (13468s, Cell Signaling Technology), Beclin1 antibody (3495, Cell Signaling Technology), P62 antibody (39749, Cell Signaling Technology), LC3 antibody (4180, Cell Signaling Technology), MMP2 antibody (ab181286, Abcam), MMP9 antibody (ab283575, Abcam), Vimentin antibody (5741S, Cell Signaling Technology), E-cadherin antibody (3195S, Cell Signaling Technology), N-cadherin antibody (13116S, Cell Signaling Technology), GAPDH antibody (ab181603, Abcam), and anti-rabbit IgG secondary antibody (ZF-0311, ZSGB-BIO) were used.

[0085] 1.16 Animal Experiments

[0086] Eight-week-old female CBA / J mice, male DBA / 2 mice, and male BALB / c mice were purchased from Beijing Biotechnology Co., Ltd., and housed in a specific pathogen-free animal facility. As previously mentioned, URSA mouse models were established using female CBA / J mice and male DBA / 2 mice, and NP mouse models were established using female CBA / J mice and male BALB / c mice. Animal experiments were conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals" (Institute of Basic Medical Sciences, Shandong Provincial Hospital Affiliated to Shandong First Medical University), and were approved by the Institutional Animal Care and Use Committee of the Institute of Basic Medical Sciences, Shandong Provincial Hospital Affiliated to Shandong First Medical University. The vaginal plugs of the mice were checked every morning.

[0087] 1.17 Fetal Absorption Rate Analysis

[0088] Significant differences exist between normal and resorbed embryos. Normal embryos are larger, fully developed, and have a red placenta, while resorbed embryos are significantly smaller and have a darker placenta. Furthermore, as mentioned earlier, the number of resorbed and viable embryos is calculated using the embryo resorption rate (R) = re / (re+F), where re is the number of resorbed embryos and F is the number of viable embryos.

[0089] 1.18 Statistical Analysis

[0090] All experiments were repeated at least three times. Values ​​are expressed as mean ± SD. Paired t-tests were used to compare data between any two groups by tests of normality and equal variance. For multiple comparisons, one-way ANOVA with Bonferroni post-hoc tests was used. Data were plotted using GraphPad Prism 8.0.1 software (GraphPad software, Inc., CA, USA). Statistical analysis was performed using SPSS software (version 16.0). Pearson correlation was used for correlation analysis. Receiver operating characteristic (ROC) curves were used to assess diagnostic values. P < 0.05 was considered significant.

[0091] 2 Experimental Results

[0092] 2.1 Upregulation of autophagy and its association with the pathogenesis of URSA

[0093] To verify whether autophagy has changed in the pathogenesis of URSA, we performed transmission electron microscopy to observe the abundance of autophagy-related granules in the villous tissue of NP and URSA patients. The results showed that the number of autophagosomes was significantly increased in URSA patients compared to the NP group, suggesting that autophagy may play a role in the pathogenesis of URSA. Figure 1 A). To further examine the involvement of autophagy and decipher the key factors involved in autophagy regulation in URSA, we examined the protein levels of key autophagy regulators, including ULK1, Beclin1, ATG13, ATG7, p62, and LC3II / I. Figure 1 B). The results confirmed that autophagy was upregulated in URSA villi, and ULK1 was the only protein among the detected targets to be elevated, suggesting that ULK1 may be the cause of enhanced autophagy. We also performed immunofluorescence (IF) assays and verified the increased protein content of ULK1 in URSA villi. Figure 1 C). We then expanded our assay for ULK1 messenger RNA (mRNA) expression in clinical samples of URSA villi, finding that ULK1 was upregulated at the mRNA level ( Figure 1 D). ROC analysis showed that ULK1 expression could serve as a diagnostic marker for URSA patients. Figure 1 E).

[0094] To functionally confirm the involvement of ULK1-mediated autophagy in the pathogenesis of URSA, we performed in vivo assays in a mouse URSA model to examine whether autophagy intervention reduced embryo resorption. Intravenous injection of chloroquine (CQ) and SBI-0206965 inhibited autophagosome-lysosome fusion and ULK1 activity, respectively, and embryo resorption rates were monitored. The study showed that both CQ and SBI-0206965 significantly reduced embryo resorption rates. Figure 1 F, G). In isolated villi of the URSA mouse model, the inhibitory effects of the two inhibitors on autophagy were verified by Western blotting of indicator markers. Figure 1 In summary, our data suggest that upregulation of autophagy is associated with the pathogenesis of URSA, and altered ULK1 transcriptional levels may be the cause of increased autophagy.

[0095] 2.2 In URSA patients, MiR-26a-5p expression was negatively correlated with ULK1 expression.

[0096] Non-coding RNAs, such as microRNAs (miRNAs) and circular RNAs, are important regulators of mRNA expression. To investigate the upstream regulatory mechanisms of ULK1, we performed microarray sequencing to screen for potential miRNAs targeting ULK1. Nineteen miRNA candidates were screened by cross-referencing differentially expressed miRNAs with a list of miRNAs retrieved from the targetscan database. Figure 2 A). To further narrow down the pool of miRNAs targeting ULK1, we used two additional databases, miRDB and DIANA. Ultimately, miR-26a-5p was the only miRNA retrieved from all these databases, and it was downregulated from our microarray analysis. Figure 2 B). Next, by expanding the clinical sample size, we confirmed that miR-26a-5p was downregulated in the villous tissue of URSA patients. Figure 2 C). Furthermore, fluorescence in situ hybridization (FISH) analysis also revealed downregulation of miR-26a-5p (C). Figure 2 D). To demonstrate the clinical significance of miR-26a-5p expression in the pathogenesis of URSA, we performed ROC analysis, which confirmed that the expression level of miR-26a-5p in chorionic villi can sensitively distinguish between URSA patients and the NP population (D). Figure 2 E). Correlation analysis clearly showed that miR-26a-5p expression was negatively correlated with ULK1 expression, strongly suggesting that miR-26a-5p may target ULK1 to determine trophoblast function. Figure 2 F).

[0097] 2.3 miR-26a-5p targets ULK1 and negatively regulates autophagy in trophoblast cells.

[0098] To further analyze the interaction between miR-26a-5p and ULK1 mRNA, we used the Targetscan database to identify two potential binding sites between miR-26a-5p and the ULK1 3' untranslated region (UTR). Figure 3 A). We designed a dual-luciferase reporter assay, subcloning the 3'UTR of ULK1 carrying the mutation site into the pGL3 plasmid. The results showed that miR-26a-5p could bind to two predicted sites on the ULK1 3'UTR (A). Figure 3 B). Simultaneously, to investigate the potential biological effects of miR-26a-5p on the trophoblast, we altered miR-26a-5p expression and measured ULK1 expression in HTR-8 / Svneo. The results showed that transfection with miR-26a-5p mimics inhibited ULK1 expression in HTR-8 / Svneo, while the inhibitor effectively enhanced its expression (B). Figure 3 (C, D). These data indicate that miR-26a-5p targets and inhibits ULK1 mRNA expression. Therefore, we further tested whether ULK1-mediated autophagy was affected by miR-26a-5p. The results consistently showed that miR26a-5p can reduce ULK1 protein abundance, thereby inhibiting autophagy levels indicated by p62 and LC3II / I protein levels. Figure 3 E). In summary, our data indicate that miR-26a-5p exerts an inhibitory effect on trophoblast autophagy through ULK1.

[0099] 2.4 Circ_0005704 interacts negatively with miR-26a-5p expression.

[0100] It has been demonstrated that circRNAs can compete with specific mRNAs for the same miRNA library. With the increasing clarity of circRNA functions and the growing feasibility of circRNA-targeted therapy, we aimed to screen circRNAs that may regulate the miR-26a-5p / ULK1 axis, providing novel interventional targets for URSA. Therefore, we performed bioinformatics analysis to retrieve all circRNAs with miR-26a-5p binding sites that intersect with the list of dysregulated circRNAs shown by microarray analysis. Figure 4 A). Twenty-four upregulated circRNAs were screened in URSA patients; these circRNAs efficiently regulate miR26a-5p expression. Figure 4B). Therefore, we used a specific miR-26a-5p probe to pull down the total RNA from the HTR-8 / Svneo lysate and detected the enrichment of all 24 circRNAs by qRT-PCR analysis. The miR-26a-5p probe significantly enriched two circRNAs (B). Figure 4 C). Subcellular localization of circRNAs is important for their functional patterns; therefore, we isolated the cytoplasm and nucleus of HTR-8 / Svneo cells and examined the distribution of these two circRNAs. The results showed that circ_0005704 was mainly located in the cytoplasm, while circ_0064954 was mainly found in the nucleus. Figure 4 D). Given that cytoplasmic circRNAs typically function via ceRNA mechanisms, we selected circ_0005704 for further analysis to investigate its regulation of the miR-26a-5p / ULK1 axis.

[0101] To further verify the interaction between circ_0005704 and miR-26a-5p, we performed RNA immunoprecipitation (RIP) and RNA pull-down experiments using the Ago2 antibody and the circ_0005704 probe, respectively. RIP analysis showed that circ_0005704 can act as an endogenous sponge for miR-26a-5p; conversely, the RNA pull-down experiment showed that circ_0005704 can bind to miR-26a-5p. Figure 4 E, F). To demonstrate that circ_0005704 indeed exists as a circular RNA, we analyzed the abundance of circ_0005704 RNA after treatment with RNase R of HTR-8 / Svneo whole RNA. The results showed that circ_0005704 was completely resistant to RNase R digestion compared to the linear transcript of the host gene C20orf94, indicating that circ_0005704 possesses a stable circular structure. Figure 4 G). Furthermore, we correlated the expression of circ_0005704 with the expression of miR-26a-5p to confirm the functional relationship between the two RNA molecules. By expanding the clinical sample, we found that circ_0005704 was upregulated in the villous tissue of URSA patients (G). Figure 4 ROC analysis confirmed that the expression of circ_0005704 could significantly distinguish URSA patients from the NP population. Figure 4 I). Pearson correlation analysis showed that the expression of circ_0005704 and miR-26a-5p was significantly negatively correlated (I). Figure 4J). Furthermore, we analyzed the nucleotide sequences that circ_0005704 can bind to miR-26a-5p, and through dual-luciferase reporter gene analysis, we further confirmed this binding (J). Figure 4 K, L).

[0102] 2.5 Circ_0005704 overexpression promotes autophagy via the miR-26a-5p / ULK1 axis

[0103] To establish the functional relationship between circ_0005704 expression and in vitro feeder cells, a plasmid overexpressing circ_0005704 was transfected into HTR-8 / Svneo cells. qRT-PCR analysis further validated the transfection efficiency and the effect of circ_0005704 on the miR-26a-5p / ULK1 axis, indicating that circ_0005704 overexpression inhibited miR-26a-5p while enhancing ULK1 expression. Figure 5 Consistently, circ_0005704 overexpression increased ULK1 protein levels and significantly promoted HTR-8 / Svneo autophagy. Figure 5 D). Furthermore, we examined whether overexpression of circ_0005704 affected trophoblast cell function. Our data showed that circ_0005704 overexpression did not affect proliferation, but inhibited HTR-8 / Svneo migration (D). Figure 5 E). The phenotype was further validated by detecting the expression of molecular biomarkers related to cell migration, including vimentin, MMP9, E-cadherin, and N-cadherin. Figure 5 F).

[0104] In animal experiments, to determine whether circ_0005704 promotes embryo resorption via miR-26a-5p, we intravenously injected plasmids overexpressing circ_0005704 with or without miR-26a-5p. We found that circ_0005704 significantly increased embryo resorption, while overexpression of miR-26a-5p decreased embryo resorption. Figure 5 G, H). Then, we isolated chorionic villus tissue from the embryo and measured the levels of autophagy and cell migration by detecting the protein levels of the aforementioned molecules. The results showed that overexpression of circ_0005704 not only enhanced autophagy but also inhibited cell migration in the chorionic villus tissue, and overexpression of miR-26a-5p could restore chorionic villus migration (G, H). Figure 5 (I, J). Therefore, we infer that circ_0005704 may inhibit cell migration via miR-26a-5p / ULK1 and lead to abortion by promoting autophagy in trophoblast cells.

[0105] 2.6 Circ_0005704 knockdown inhibits autophagy and reduces embryonic resorption via miR-26a-5p / ULK1.

[0106] To further investigate the functional relationship between circ_0005704 and trophoblast cells, HTR-8 / Svneo cells were initially transfected with small interfering RNA (siRNA) targeting circ_0005704. Subsequently, qRT-PCR analysis was used to verify the transfection efficiency of circ_0005704 and its effect on the miR-26a-5p / ULK1 axis. The results showed that circ_0005704 knockdown promoted miR-26a-5p and reduced ULK1 expression. Figure 6 Furthermore, after circ_0005704 knockout, a significant decrease in ULK1 protein levels and inhibition of autophagy were observed in HTR-8 / Svneo. Figure 6 D). Furthermore, the effect of circ_0005704 knockout on the functional properties of trophoblast cells was investigated. The results showed that although circ_0005704 knockout did not affect cell proliferation, it inhibited cell migration in HTR-8 / Svneo. Figure 6 E). This observation is supported by an assessment of relevant molecular biomarkers associated with cell migration, including vimentin, MMP9, E-cadherin, and N-cadherin (E). Figure 6 F).

[0107] From an in vivo perspective, the potential mediating role of circ_0005704 in promoting embryo resorption via miR-26a-5p was further explored. To address this issue, intravenous injection of siRNA targeting circ_0005704 was performed, regardless of the use of miR-26a-5p inhibitors. Results showed that circ_0005704 knockdown induced a significant decrease in embryo resorption, while miR-26a-5p inhibition counteracted this effect. Figure 6 G, H). Subsequently, villous tissue was isolated from the embryo to assess autophagy and cell migration-related proteins. The results showed that knockout of circ_0005704 not only inhibited autophagy but also enhanced cell migration in the villous tissue, and inhibition of miR-26a-5p mitigated this effect. Figure 6 Based on these findings, it is speculated that circ_0005704 knockout may promote cell migration through the miR-26a-5p / ULK1 axis and may prevent miscarriage by reducing autophagy in trophoblast cells.

[0108] In summary, the data provided by this invention indicate that circ_0005704 promotes autophagy in trophoblast cells, thereby determining migration and miscarriage rates in URSA patients. Overexpression of circ_0005704 eliminates the inhibitory effect of miR-26a-5p on ULK1, leading to its accumulation and enhanced autophagy, which in turn affects trophoblast cell migration. Mechanistically, circ_0005704 acts as a molecular sponge for miR-26a-5p, thereby attenuating the binding of miR-26a-5p to the 3'UTR of ULK1 mRNA. Importantly, upregulation of circ_0005704 is closely associated with downregulation of miR-26a-5p and is positively correlated with ULK1-mediated autophagy in patients and URSA models. In conclusion, these results reveal a regulatory axis of ULK1-mediated autophagy that promotes the pathogenesis of URSA induced by dysregulation of circ_0005704 and miR-26a-5p.

[0109] Matters not covered in this invention are common knowledge.

[0110] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. Application of substances that detect the expression levels of circ_0005704 and related biological elements in the preparation of products for screening, diagnosis, detection, monitoring, or prognosis of unexplained recurrent spontaneous abortion.

2. The application as described in claim 1, characterized in that, The bio-elements associated with circ_0005704 include miR-26a-5p and ULK1.

3. The application as described in claim 1, characterized in that, The substances used to detect the expression levels of circ_0005704 and its related biological elements include reagents for detecting the expression levels of circ_0005704, miR-26a-5p, and ULK1 encoding genes based on real-time quantitative PCR, in situ hybridization, gene chips, and gene sequencing, and / or reagents for detecting the expression level of ULK1 protein based on immunoassay methods.

4. The application as described in claim 1, characterized in that, The products include primers, probes, (gene or protein) chips, nucleic acid membrane strips, detection kits, detection devices, and detection equipment for detecting the expression levels of circ_0005704 and its related biological elements.

5. A system for screening, diagnosing, detecting, monitoring, or prognosing unexplained recurrent spontaneous abortion, characterized in that, The system includes: The acquisition unit is configured to acquire the expression levels of circ_0005704 and its related biological elements in the subject's test sample; The assessment unit is configured to assess and determine the subject's disease status based on the subject's circ_0005704 and its related bio-elements obtained by the acquisition unit.

6. The system as described in claim 5, characterized in that, The bio-elements associated with circ_0005704 include miR-26a-5p and ULK1.

7. The system as described in claim 5, characterized in that, The test samples include blood samples (including peripheral blood mononuclear cells) and chorionic villus tissue samples from the subject.

8. The application of circ_0005704 and its related biological elements as targets in the preparation and / or screening of drugs for unexplained recurrent spontaneous abortion; in, The bio-elements associated with circ_0005704 include miR-26a-5p and ULK1.

9. The use of substances that inhibit circ_0005704 expression or reduce its activity in any one or more of the following: a) Promote miR-26a-5p expression or prepare products that promote miR-26a-5p expression; b) Inhibit ULK1 expression or prepare products that inhibit ULK1 expression; c) Promote the migration of trophoblast cells or prepare products that promote the migration of trophoblast cells; d) Inhibit autophagy in trophoblast cells or prepare products that inhibit autophagy in trophoblast cells; g) Products for the prevention and / or treatment of unexplained recurrent spontaneous abortion.

10. The application as described in claim 9, characterized in that, Substances that inhibit circ_0005704 expression or reduce its activity include RNA interference molecules or antisense oligonucleotides targeting circ_0005704, small molecule inhibitors, siRNA, shRNA, and substances that induce lentiviral infection or gene knockout.

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